A transcription initiation factor for transcription of ribosomal RNA in Acanthamoeba, castellanii by Pol I has been purified. This factor, TIF-IB, appears to be a complex of approximately 450 kD. Site-specific cross-linking experiments suggested that there could be two factors bound to the promoter rather than one. A 236 bp piece of DNA containing the promoter was incubated with TIF-IB and examined in the STEM. The majority of the protein-DNA complexes had the protein factor approximately in the middle of the DNA fragment. The mass of the protein was measured and was approximately 500 kD, consistent with the TIF-IB binding as a monomer. Studies involving the addition of Pol I to TIF-IB on DNA restriction fragments containing the promoter with point mutations to stall the transcribing polymerase have been initiated. Pol I appears morphologically quite different from TIF-IB although its mass as also about 500 kD. These experiments are just beginning.